Mammalian thioredoxin reductases are pyridine nucleotide-disulfide oxidoreductases that contain the unusual amino acid selenocysteine. In vivo, selenocysteine is coded in the mRNA by a UGA codon (normally a stop codon). This makes expression of these proteins in heterologous systems difficult. A second remarkable feature of the enzyme is that it utilizes a Se-S bond between adjacent residues in the catalytic cycle. Formation of this bond results in an 8-membered ring structure, and requires that the intervening peptide bond between neighboring cysteine and selenocysteine residues adopt a cis configuration. It is the major hypothesis of this proposal that the enzyme uses the catalytic power of adjacent cysteine and selenocysteine residues in a cis configuration to catalyze reduction of target disulfides. The cis configuration is expected to be more reducing because of the high local concentration of thiol this geometry imposes on the active-site. This proposal utilizes a semisynthetic system for studying the enzyme mechanism. This system divides the protein into two modules, one protein module, and a synthetic peptide containing selenocysteine. This semisynthetic approach to studying thioredoxin reductase and other selenocysteine-containing proteins is novel and unique. This semisynthetic system can be used to generate the wild-type protein and to insert peptide bond isosteres that restrict the geometry of the peptide bond. These isosteres allow for the study of the enzyme mechanism in great detail. The semisynthetic system also permits structure-function studies to be explored by using the method of peptide complementation. Model disulfide compounds that form an 8-membered ring will be synthesized. The redox potentials of these model compounds will be correlated to their backbone geometry. The redox potential of the catalytic disulfide bond of TR (S replaces Se) will be measured and correlated to the model compounds to determine the geometry of the peptide bond in the enzyme active-site. The importance of the thioredoxin system in disease processes such as cancer, arthritis, and malaria gives impetus to the study of the enzyme mechanism for the development of potential therapeutic inhibitors.